NEW TOOLS FOR EXPLORING THE DYNAMIC INTERACTOME

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 描述（由申请人提供）：基因组革命已经被确定了大量遗传信息的技术所授权。虽然核酸编码这些信息，但蛋白质对其起作用。蛋白质在丰度和性质上具有令人难以置信的多样性，使其在手头的动态任务中具有高度的通用性，但同时也非常难以分析。正是由于这些原因，蛋白质组学革命仍然落后于基因组学革命。事实上，对细胞中蛋白质动态特性的全面分析在很大程度上仍然超出了目前的能力。在这里，我们寻求革命性的蛋白质组学的协同相结合的改进，在现有的技术与新的方法。我们将克服蛋白质组学技术三个关键领域的主要瓶颈。首先，我们将改革生产阶段，以产生完整的大分子复合物，这样我们就能够在可视化其在细胞中的位置的瞬间内将标记的大分子复合物冻结在适当的位置，然后将其与其所有组分和邻居一起分离。其次，我们将优化每个复合物的分析，以便其大分子组成，结构和动力学将被量化和分析。第三，我们将开发软件来整合我们的数据，并以前所未有的细节来表示许多动态亚细胞组装中大分子参与者的行为。我们将寻求通过在4个实验系统中进行beta测试，使这些技术快速，稳健和常规。这些系统专注于遗传信息途径的各个方面，因为（i）这是真核生物的核心，（ii）它将使我们能够开发技术来分析所有3种携带信息的生物大分子（DMA，RNA和蛋白质）的相互作用。首先，我们将沿着一大片染色质走，确定结构蛋白和调节因子的正常流动，这些蛋白和调节因子共同组成了基因组的动态片段。其次，我们将跟踪RNA转录后的过程，因为它是加工，包装和从细胞核输出的;我们将列举在其成熟过程中参与每种RNA分子的蛋白质。最后，我们将揭示两种致病性人类病毒，HIV和CMV，如何颠覆宿主的遗传信息通路并用自己的遗传信息通路取而代之。通过创建一个国家动态相互作用研究中心，我们将耦合一个既定的质谱资源，细胞生物学实验室，系统生物学资源和计算生物学中心。作为更大的NIH路线图的一部分，该中心的目标将是创建新的和有用的工具来阐明大分子相互作用的动力学。总之，本提案寻求支持，以推动我们的方法进入全新的领域，并在生物医学界传播这些方法。该中心将使社区能够组装细胞中相互作用的各种详细，动态表示，这将有助于阐明所有细胞过程的基本原则，从而弥合功能基因组学，蛋白质组学和系统生物学之间的差距。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): The genomic revolution has been empowered by technologies that have determined a vast pool of genetic information. While nucleic acids encode this information, it is the proteins that act on it. Proteins are incredibly diverse in their abundance & their properties, making them highly versatile for the dynamic tasks at hand but at the same time exceptionally difficult to analyze. It is for these reasons that the proteomic revolution still lags behind the genomic revolution. Indeed, the comprehensive analysis of the dynamic properties of proteins in cells is still largely beyond current capabilities. Here, we seek to revolutionize proteomics by synergistically combining improvements in established techniques with new approaches. We will overcome major bottlenecks in 3 key areas of proteomics technology. First, we will reform the production stage for generating intact macromolecular complexes, so that we will be able to freeze a tagged macromolecular complex in place, within moments of visualizing its position in the cell, & then isolate it together with all its components & neighbors. Second, we will optimize the analysis of each complex such that its macromolecular composition, structure, & dynamics will be quantified & analyzed. Third, we will develop software to integrate our data & represent in unprecedented detail the actions of the macromolecular players in many dynamic subcellular assemblies. We will seek to make these techniques rapid, robust & routine by beta testing them in 4 experimental systems. These systems focus on aspects of the genetic information pathway, because (i) this is core to eukaryotes, & (ii) it will allow us to develop techniques to analyze the interactions of all 3 information-carrying biological macromolecules (DMA, RNA & proteins). First, we will walk along great stretches of chromatin, determining the normal flux of structural proteins & regulatory factors that together comprise dynamic segments of the genome. Second, we will follow the course of RNA after transcription, as it is processed, packaged & exported from the nucleus; we will enumerate the proteins that dance attendance on each kind of RNA molecule during its maturation. Finally, we will expose how 2 pathogenic human viruses, HIV & CMV, subvert their host's genetic information pathway & supplant it with their own. By creating a National Center for Dynamic Interactome Research, we will be coupling an established mass spectrometry resource, cell biology laboratories, a systems biology resource, & a computational biology center. As part of the larger NIH roadmap, the center's aim will be to create new & useful tools to elucidate the dynamics of macromolecular interactions. In summary, the present proposal seeks the support to advance our methods into totally new areas, & to spread these methods amongst the biomedical community. The Center will enable the community to assemble the kinds of detailed, dynamic representations of the interactions in the cell that will help elucidate the principles underlying all cellular processes, thus bridging the gaps between functional genomics, proteomics, & systems biology.